JP2019533710A - Attenuation of bacterial toxicity by attenuation of bacterial folate transport - Google Patents

Abstract

The present invention relates to bacterial infections, vaccines against such infections and bacterial vaccines. More particularly, the present invention relates to a vaccine against streptococcal infection in pigs. Provided is a bacterial ΔFolT mutant having a reduced ability to transport folic acid, wherein the ability is reduced by functionally deleting the folate transporter (FolT) function. Further provided is a method of reducing bacterial toxicity comprising reducing the ability of a bacterium to transport folic acid.

Description

  The present application relates to bacterial strains, drugs against bacterial infections, and bacterial vaccines. More particularly, it relates to a vaccine against Streptococcus infection in pigs.

  Plants, fungi, certain protists, and many bacteria produce new folic acid (vitamin B9) from GTP and chorismate, but higher animals lack key enzymes that are key to the synthetic pathway Therefore, dietary folic acid is required. Folic acid is important for health and folic acid antagonists are widely used in cancer chemotherapy and as antibacterial agents. For this reason, folate synthesis and salvage pathways have been widely characterized in model organisms, and both bacterial and plant folate synthesis pathways have been modified to enhance food folate content. Tetrahydrofolic acid is an essential cofactor for many biosynthetic enzymes. This acts as a one-carbon unit carrier in the synthesis of important metabolites such as methionine, purine, glycine, pantothenic acid, and thymidylic acid. For example, the enzyme encoded by the panB gene, ketopantoate hydroxymethyltransferase, requires a tetrahydrofolate cofactor for the synthesis of pantothenic acid precursors. Since tetrahydrofolic acid is newly synthesized in bacteria, inhibition of its synthesis results in cell death. In fact, two very effective antibiotics, sulfonamides and trimethoprim, kill bacterial cells by blocking the production of tetrahydrofolic acid. Two such antibiotics, often used in combination with each other, are generally prescribed for the treatment of urinary tract infections, intestinal infections such as bacterial dysentery, and respiratory tract infections. The success of such agents means that many pathogens are vulnerable to tetrahydrofolate synthesis inhibitors. Bacteria have a multistep pathway for the synthesis of tetrahydrofolate cofactor. In one division of this pathway, the metabolites, chorismate and glutamine are substrates for aminodeoxychorismate synthase encoded by the B. subtilis genes pabA and pabB, which are 4-amino-4 Produce deoxychorismic acid. The aminodeoxychorismate lyase encoded by Bacillus subtilis pabC then converts 4-amino-4-deoxychorismate to paraaminobenzoic acid (PABA), an important precursor. In another sector, some of the enzymes, including those encoded by Bacillus subtilis mtrA, folA, and folK, are the precursors 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydrokisopteridine dilin Produce acid. This precursor and PABA are substrates for dihydroprotein acid synthetase encoded by the Bacillus subtilis sul gene (homologous to the E. coli dhps and folP genes), which produces dihydroprotein acid. Sulfonamides such as sulfamethoxazole are competitive inhibitors of dihydroprotein acid synthase. Dihydroprotein acid is modified by a bifunctional enzyme encoded by Bacillus subtilis folC to produce dihydrofolic acid. Eventually, DHFR (dihydrofolate reductase) encoded by Bacillus subtilis dfrA modifies this dihydrofolate to produce the final product, tetrahydrofolate. Trimethoprim is a competitive inhibitor of bacterial DHFR. This selectivity is important because eukaryotic DHFR is not disturbed by antibiotics. Folic acid is almost certainly essential for all sequenced bacteria except M. hyopneumoniae. However, not all bacteria synthesize folic acid de novo and instead rely on external supplies. To predict the lack of a new synthetic pathway, HPPK (FolK) and DHPS (FolP) proteins are used as signature proteins. Many bacteria lack the homologues of both such genes and are therefore almost certainly dependent on the reduction and glutamylation of intact folate taken from the environment. These are mainly host-associated bacteria such as Mycoplasma or Treponema or organisms that inhabit folate-rich environments such as Lactobacillus.

  Streptococcus species have a wide range of species that cause infections in livestock and humans and are often classified according to the Lancefield group. In particular, Lancefield classifies based on serological determinants or antigens present in the bacterial capsule, allowing only approximate determination. Bacteria from different groups often show cross-reactivity with each other, but other streptococci cannot assign group determinants at all. Many groups are further differentiable based on serotyping, and such serotypes further contribute to the broad antigen diversity of streptococci. In fact, this creates a number of difficulties in the diagnosis of streptococcal infection and in vaccination against streptococcal infection. Lancefield Group A Streptococcus (GAS, Streptococcus pyogenes) is common in children and causes nasopharyngeal infections and their complications. Group B Streptococcus (GBS) is a leading cause of bacterial sepsis and meningitis in human neonates and has emerged as a significant pathogen in neonates in developing countries. Lancefield Group B Streptococcus (GBS) has also been found associated with cattle and causes mastitis. Infections caused by Lancefield group C infections, such as Streptococcus (S. equi), Streptococcus zooepidemicus, S. dysgalactiae et al., Mainly in horses, cattle and pigs. It can be seen. Lancefield Group D (S. bovis) infections are found in all mammals and some birds and can cause endocarditis or sepsis. Lancefield E, G, L, P, U, and V groups (Streptococcus porcinus, Streptococcus canis, Streptococcus disgalactie) are found in various hosts and are neonatal infections Causes infectious diseases, nasopharyngeal infections or mastitis. In Lancefield R, S, and T groups (and non-classified types), S. suis is seen, which is an important cause of meningitis, sepsis, arthritis and sudden death in young pigs is there. This can also cause meningitis accidentally in humans. Non-classified streptococcal species such as S. mutans causing caries in humans, Streptococcus uberis causing mastitis in cattle, and invasive diseases such as pneumonia, bacteremia And S. pneumonia that cause meningitis.

Streptococcus pneumoniae is a zoonotic pathogen and is ubiquitously present in the swine population in the pig farming industry. Thirty-three capsular serotypes have been reported to date ^[1] , and serotypes ¹ , 2, 7, 9 and 14 have been isolated from affected pigs in Europe ^[2] . The virulence of the strains varies between serotypes and between serotypes, and in serotype 2, toxic, non-toxic and weakly toxic isolates have been isolated, which is a toxicity marker, muramidase releasing protein (MRP) And extracellular factor (EF) ^[3] as well as the expression of slicin ^{[4, 5]} . Streptococcus pneumoniae in adult pigs is asymptomatic, but in young piglets this nasopharyngeal carriage increases susceptibility to Streptococcus pneumonia invasive disease, causing meningitis, arthritis and serositis As well as high mortality rates. In Western countries, humans who are occupationally in contact with pigs or raw pork can also be infected by Streptococcus pyogenes, but this incidence is very low. When humans are infected with invasive Streptococcus pneumoniae, they develop clinical signs similar to pigs, and patients often suffer from hearing loss after recovery. ^[6] In Southeast Asia, particularly Serotype 2 Streptococcus is considered an emerging pathogen for humans and is recognized as a major cause of bacterial meningitis ^[7-10] . In Southeast Asia, clinical signs of Streptococcus pneumoniae human infections have been reported to be more severe compared to other parts of the world, and patients were diagnosed with toxic shock-like syndrome, sepsis and meningitis Develops. Little is known about the pathogenesis of diseases caused by Streptococcus pneumoniae. Various bacterial components, such as extracellular membranes and cell membrane-related proteins, play a role in pathogenesis. Furthermore, the coating has been shown to be an important virulence factor by making such microorganisms resistant to phagocytosis. Current methods of preventing Streptococcus pneumoniae infections in pigs include antibiotic therapy in affected pigs combined with vaccination with self-vaccines ^[11] . Self-vaccination with bacterin vaccine against serotype 2 has been shown to be able to reduce clinical outbreaks on the farm, but this is not the case with serotype 9, in which case It cannot be considered effective defense ^{[12, 13]} . In addition to the low effectiveness of bacterin vaccines against serotype 9 infections, they can only protect against serotypes present in the vaccine. However, as mentioned above, autologous vaccines have become a temporary solution to clinical outbreaks because some serotypes can cause disease. As a long-term solution to Streptococcus pyogenes infections, vaccines that protect extensively from all (pathogenic) serotypes are needed. Although much research has been done to find suitable vaccine candidates, no cross-protectable vaccine has yet been provided.

  Selecting a parent bacterial strain that is generally capable of folate transport and folate synthesis, and mutations, deletions or insertions in the DNA region encoding the bacterial folate substrate binding protein (known as the foT gene in Streptococcus pyogenes) Select bacteria from this parent strain for modification, and select bacteria with the ability to grow to a similar rate as the parent strain in vitro but to a lower rate compared to the parent strain in vivo A method of producing bacteria, preferably for use in a vaccine, preferably for use in a vaccine that provides protection against bacterial infections. Selecting a parent bacterial strain that is generally capable of folate transport and folate synthesis, and preferably by recombination means, in the DNA region encoding the bacterial folate substrate binding protein (known as the folT gene in Streptococcus pyogenes) By transforming the bacterium from this parental strain by modifying the bacterium, such as deletion or insertion, and in vitro it grows to the same rate as the parental strain, but in vivo compared to the parental strain. Selecting a bacterium having the ability to grow to a low rate, preferably further providing a method for producing a bacterium for use in a cutin, preferably for use in a vaccine that provides protection from bacterial infections . Further provided is a bacterium or bacterial culture obtainable or obtained by the selection or transformation method of the present invention. The bacteria provided herein are preferably categorizable as Firmicutes, preferably streptococci, more preferably swine streptococci. Further provided is a composition comprising a bacterium or bacterial culture that is capable of growing to a similar rate as the parental strain in vitro but grows in vivo to a lower rate compared to the parental strain. Further provided is the use of such a composition for the production of a vaccine. Preferably, such a vaccine comprises a bacterium or bacterial culture that can grow to a similar rate in vitro as the parental strain, but grows in vivo to a lower rate compared to the parental strain.

  Further provided is a method for reducing (attenuating) the toxicity of a bacterium, preferably a bacterium capable of de novo folate synthesis, comprising reducing the ability of the bacterium to transport folic acid. Provided herein is a bacterium commonly referred to as a ΔFolT mutant, in particular a porcine streptococcal strain whose ability is strongly reduced by functionally deleting folate transporter (FolT) function. Provided herein. This bacterium, as provided herein, still has the ability to produce folic acid for its own use by applying its novel folate synthesis pathway. If such synthetic pathways remain intact, their ability to grow in vitro remains unaffected, but surprisingly, their growth and toxicity in the host (in vivo) is strongly reduced. Is shown. Such bacterial strains grow well in vitro but do not grow as well as their parent strain in vivo and are associated with a strong reduction in toxicity in vivo and are very useful as vaccine strains. In such strains or mutants provided herein, on the one hand, they are essentially unaffected in folic acid synthesis and can therefore grow to high titers and are therefore relatively easy and cheap to produce, On the other hand, since the growth in the host is reduced and the toxicity is reduced compared to the parent strain, it is relatively harmless after in vivo application and is extremely useful as a vaccine against bacterial infections.

  Modified in the DNA region encoding the folate substrate binding protein (known as the folT gene in Streptococcus pyogenes) and has a growth rate similar to the parental strain in the medium (in vitro), but in vivo Prototype ΔFolT mutant having a low growth rate was deposited as “CBS140425 Streptococcus pneumoniae ΔFolT mutant” on August 19, 2015 for the purpose of a patent procedure based on the laws of the Budapest Treaty at the Centralbureau voor Schimmel cultures. ing. Modified in the DNA region encoding the folate substrate binding protein (known as the folT gene in Streptococcus pyogenes) and has a growth rate similar to the parental strain in the medium (in vitro), but in vivo Another prototype ΔFolT mutant with a low growth rate has been deposited on August 25, 2017 at the Westerdikk Fungal Biodiversity Institute as “CBS143192 Streptococcus pneumoniae ΔFolT2 mutant”.

  The ability of bacteria to synthesize new folic acid can be determined by methods known in the art, for example, by testing bacterial growth in culture medium without folic acid compared to culture medium with folic acid, or by BMC Genomics 2007 8: 245 (doi: IO.1186 / 1471-2164-8-245, incorporated herein by reference) and can be readily tested by other methods. Most bacteria have at least two independent pathways for acquiring tetrahydrofolate, one following the classical folate synthesis pathway and one following a rapid method using a folate transporter that imports folic acid . In vitro, it is presented here that there are sufficient nutrients and energy available using classical synthetic pathways. While not wishing to be bound by theory, providing a possible explanation for the actions found by the inventors, in vivo, the lack of nutrients, and therefore lack of energy, can lead to the generation of THF by the classical pathway. Investing can be much more difficult. Therefore, it is conceivable to select an alternative method for transferring folic acid. Based on ongoing experiments, we hypothesize that the expression of folT is burdensome for bacteria, probably due to its high hydrophobicity. In vitro, the growth rate decreases when the expression of folT increases. This is probably because the expression of folT is very tightly controlled by the presence of the riboswitch. folT should be expressed only when absolutely necessary. In summary, there appears to be a balance between nutrient availability and the requirements for THF and the burden of protein expression. It is found here by the inventors in the present invention that tilting this balance to one side in vitro increases novel folate synthesis and tilting to the other side in vivo increases folate transport. Surprisingly, knocking out folate transport in the in vivo pathway by attenuating (decreasing) folate transport in the in vivo pathway, preferably by functionally deleting the folate transporter function, results in bacteria in the host and not in the medium. Toxicity is reduced. In a preferred embodiment, a ΔFolT mutant of bacteria having a reduced ability to transport folic acid, wherein this ability is reduced by functionally deleting folate transporter (FolT) function. provide. Specifically, mutations, deletions or insertions are made in the bacterial folT gene or in the promoter of the gene, in particular, attenuated (reduced) bacterial folate substrate binding protein (FolT) expression and / or function. A method is provided herein. Such mutations, deletions or insertions can be detected by PCR and / or sequence analysis as is known in the art. In a particular embodiment of the invention, the folT gene is knocked out to greatly attenuate bacteria such as Streptococcus pyogenes and is suitable for use in vivo as a vaccine strain that can also be easily cultured in vitro. To provide a method. In another embodiment, the folate substrate binding protein FolT is preferably maintained while the FolT immunogenicity remains essentially intact, and most preferably the hydrophilic protein domain of FolT remains essentially intact. Methods are provided for reducing toxicity by mutating, deleting, or inserting into bacterial DNA encoding a transmembrane region. In another embodiment, in FolT encoding a bacterial DNA region encoding a region in Streptococcus pneumoniae characterized by a peptide domain having a stretch of amino acids FYRKP (SEQ ID NO: 16), a region important for substrate binding. Methods are provided for reducing toxicity by making mutations, deletions or insertions. Preferably, arginine (R) is mutated at least in the stretch of FYRKP (SEQ ID NO: 16) to eliminate folate binding. In a preferred method of the invention, the bacteria can be classified as Pharmicutes, preferably streptococci, more preferably swine streptococci. The ΔFolT mutant of the present invention preferably has the ability to synthesize folic acid. If such a synthetic pathway is left intact, the ability to grow in vitro (in the medium) remains unaffected. , The toxicity in the host (in vivo) is strongly reduced, making it very suitable for use in vaccines. The ΔFolT mutation of the present invention is characterized by reduced expression of FolT (decreased or disrupted), eg, reduced expression of FolT itself or reduced expression of FolT mutant protein, For example, it is preferred that the mutant can be tested with a FolT-specific nucleotide construct in the in vitro transcription / translation test described in the experimental section herein. In one particular preferred embodiment, the ΔFolT variant of the present invention, deposited on 19th August 2015 at the Centralbureau au Schimmelcultures as “CBS140425 Streptococcus pneumoniae ΔFolT variant” is provided. In another particular preferred embodiment, the ΔFolT variant of the present invention, deposited at the Westerdjk Fungal Biodiversity Institute on August 25, 2017 as “CBS 143192 Streptococcus ΔFolT2 variant” is provided.

  In another particular preferred embodiment, a ΔFolT mutant of the present invention is provided. To prepare a ΔFolT mutant nucleotide construct as a control construct in expression studies with additional bacterial ΔFolT mutants, any of these deposits can be used to perform FolT mutant gene expression or FolT mutant protein expression. You can also test. Any such deposit may be used to provide a ΔFolT mutant bacterial culture of the invention, or a composition comprising a ΔFolT mutant bacterial culture. Further provided are bacteria with reduced toxicity obtained or obtained with the methods provided herein, and cultures of such bacteria. Further provided are compositions comprising the ΔFolT mutant bacteria or ΔFolT mutant cultures of the present invention, and the use of such compositions for the production of vaccines. Further provided is a vaccine comprising a ΔFolT mutant bacterium or a ΔFolT mutant culture provided herein. In a preferred embodiment, a streptococcal vaccine strain or vaccine is provided comprising a ΔFolT mutant capable of expressing non-streptococcal proteins. Such a vector streptococcal ΔFolT mutant vaccine strain enables protection from pathogens other than streptococci when used in vaccines. Because of its non-toxic properties, the streptococcal vaccine strain or ΔFolT mutant provided herein is specific and persistent not only against streptococcal antigens, but also against other antigens expressed by this strain. To produce a positive immune response. In particular, an antigen from another pathogen is expressed here without the adverse effects of an antigen or pathogen that would otherwise be harmful to the host. Examples of such vectors are streptococcal vaccine strains or ΔFolT mutant strains, in which the antigen is actinobacillus pleuropneumonia, Bordetella, Pasteurella, E. coli, Salmonella (Salmonella), Campylobacter, Serpurina, and other pathogens. Further provided is a vaccine comprising a streptococcal vaccine strain or ΔFolT variant and a pharmaceutically acceptable carrier or adjuvant. Carriers or adjuvants are well known in the art, and examples include phosphate buffered saline, physiological saline, oil-in-water (water-in-water) emulsions, aluminum hydroxide, Specol, block polymers or copolymers and the like. The vaccines of the present invention can include vaccine strains in either killed or live form. For example, killed vaccines containing strains that (over) expressed virulence factors such as streptococci or heterologous antigens are very suitable for eliciting an immune response. In certain embodiments, the non-toxic properties provide a vaccine in which the strain is alive, and streptococcal vaccine strains based on the ΔFolT mutant provided herein produce a specific and sustained immune response Fits to There is further provided a method of controlling or eradicating streptococcal disease in a population, comprising vaccinating a subject of the population with a ΔFolT mutant vaccine of the present invention. It is presented herein that Streptococcus pneumoniae has an operon that has an important role in the pathogenesis and / or toxicity of Streptococcus pyogenes. The operon encodes two genes that are involved in folic acid acquisition and processing of folic acid into tetrahydrofolate. Folic acid is a general term for the water-soluble vitamin B group, where folic acid refers to various tetrahydrofolic acid derivatives. Such derivatives can enter the main folate metabolic cycle either directly or after initial reduction and methylation to tetrahydrofolate. Folic acid is essential for all living organisms, both prokaryotes and eukaryotes, making folate metabolism an important process. The folT-folC operon is believed to form an escape pathway and acquire folic acid under folate-restricted conditions, for example in vivo, so that the host sequesters folic acid for its own use. Under such conditions, the expression of the folT-folC operon is induced by a riboswitch. When folic acid levels drop, the riboswitch is opened and tetrahydrofolic acid is released therefrom. This allows initiation of translation by release of the ribosome binding site. The expression of folT-folC allows Streptococcus pyogenes to directly transfer folic acid by the folate transport complex, and subsequently process folic acid to tetrahydrofolate by folC. Because folic acid is important for nucleotide synthesis, acquiring folic acid directly affects the growth rate of Streptococcus pyogenes. Reduction in in vivo growth rate results in reduced toxicity. This finding was further supported by demonstrating that a folT syngeneic knockout mutant such as CBS 140425 or the mutant deposited as CBS 143192 is strongly attenuated and useful as a vaccine. Operons are actually involved in in vivo pathogenesis. The invention described herein provides herein compositions and vaccines comprising such mutants and cultures, and such FolT knockout mutants with reduced toxicity. Further provided are immunogenic compositions comprising such reduced toxicity bacteria, and the use of such compositions for the production of vaccines. Further provided herein is a vaccine comprising a mutant strain deposited as a ΔFolT mutant bacterial strain, eg, CBS140425, or CBS143192, or a culture or composition thereof. A dispenser for administering the vaccine to an animal, preferably a pig, and a ΔFolT mutant, for example CBS140425, or a mutant of the invention deposited as CBS143192, or a culture or composition thereof, optionally with instructions for use Further provided is a kit for vaccination of an animal, preferably a pig, against a disease associated with a Streptococcus pyogenes infection, comprising: In conclusion, a general method for reducing bacterial toxicity, including the step of reducing the ability of the bacterium to transport folic acid, in particular, an applicable method in which the bacterium has the ability to synthesize new folic acid. provide. The method of the invention comprises the steps of selecting a bacterium having a functional folate substrate binding protein and then attenuating the expression and / or function of the bacterial folate substrate binding protein (FolT), in particular Reducing the toxicity by applying mutation, deletion or insertion in the bacterial folT gene, for example, by applying mutation, deletion or insertion in the bacterial DNA encoding the promoter of the folT gene. In certain embodiments of the invention, it is preferred to attenuate the toxicity by mutating, deleting or inserting into the bacterial DNA encoding the transmembrane region of the folate substrate binding protein FolT. In another particular embodiment, the toxicity is attenuated by making mutations, deletions or insertions in FolT encoding a bacterial DNA region that encodes a region important for substrate binding. Furthermore, a method for obtaining an immunogenic composition or vaccine, wherein the bacterium is Fermicutes, preferably Streptococcus, more preferably Streptococcus pneumoniae, more preferably “CBS140425 Streptococcus pneumoniae ΔFolT mutant” as a central bureauau floor The ΔFolT mutant strain deposited at Schimmelcultures on August 19, 2015, and most preferably the ΔFolT mutant strain deposited at the Westerdjk Fungal Biodiversity Institute on August 25, 2017 as “CBS143192 Streptococcus pneumoniae ΔFolT2 mutant” Certain methods applicable to bacteria are provided herein. Further provided is a bacterium with reduced toxicity obtained or attenuated by the method of the present invention that attenuates toxicity by attenuating FolT expression, a culture of such bacteria, and reduced toxicity Compositions comprising such bacteria or cultures of such bacteria having reduced toxicity, and immunogenic compositions comprising bacteria having reduced toxicity or cultures of bacteria having reduced toxicity are provided, wherein FolT expression is provided The use of attenuated bacterial cultures or compositions of bacterial cultures with reduced FolT expression for the production of vaccines is provided. Further provided is a vaccine comprising a bacterium with attenuated FolT expression or a culture of bacteria with attenuated FolT expression, or comprising a composition of a bacterial culture with attenuated FolT expression. a) a dispenser for administering a vaccine to an animal; and b) a specific bacterial syngeneic knockout strain (mutant) with attenuated FolT expression or a culture of a specific bacterial syngeneic knockout strain with attenuated FolT expression. Or a specific bacterium having the ability to infect FolT expression, comprising a composition comprising a culture of a cognate knockout strain of a particular bacterium with attenuated FolT expression, and optionally c) instructions for use Further provided are kits for vaccination of animals against related diseases. Such a specific bacterium of the present invention that has been subjected to attenuation of FolT expression and reduced ability to transport folic acid, and this ability is reduced by functionally deleting folate transporter (FolT) function The bacteria generally have good growth properties in the culture medium, especially when using the ΔFolT mutants of the present invention that have the ability to synthesize folic acid, leaving such synthetic pathways intact. Then, the ability to grow in vitro (in the medium) remains unaffected, but the toxicity in the host (in vivo) is strongly reduced, making it very suitable for use in vaccines.

Use complementarity test method where V [10] contains two incomplete ORFs (gray blue arrows) and a putative operon (purple) with orf2 [10] and foC [10] preceded by the operon's putative promoter (Clearly, only the sequence of the -35 region (TGGACA) of the putative promoter is shown in the figure). A construct containing either orf2 [10] or folC [10] (purple) preceded by a predicted -35 region (purple) of the putative promoter region of the operon was generated. A construct was generated containing orf2 (green) from strain S735 with the -35 region (TGGTCA) (green) of the putative S735 promoter. The construct was mutagenized to contain the -35 region (TGGACA) (purple) of the putative promoter sequence of strain 10 to obtain orf2 [S735] [t488a]. FIG. 5 shows in vitro transcription / translation of FolC and ORF2 / FolT. In vitro transcription translation of the control construct pCOM1 (lane 1) and the constructs pCOM1-V [10] (lane 2) and pCOM1-folc [10] (lane 3). Molecular weight markers are shown on the right side in kDa. FolC expression was detected at a predicted amount of 46.8 kDa (closed arrowhead), while OR2 / FolT expression was detected at a molecular weight of 14 kDa (open arrowhead) lower than the predicted amount (20.5 kDa). FIG. 6 shows a predicted riboswitch for tetrahydrofolic acid using Rfam. The three-dimensional structure of RNA showed that the two putative ribosome binding sites (blue arrows) are riboswitches that are difficult to access because they are folded. FIG. 6 shows Clustal W alignment of various FolT sequences. * Indicates the same amino acid;: indicates conservability between groups with strong similarity; Indicates conservation between groups with weak similarity. CB = Clostridium bolteae (SEQ ID NO: 17), CP = Clostridium phytofermentans (SEQ ID NO: 18), AM = Alkalifilus metalligendi (19) TT = Thermoanaerobacter tengcongensis (SEQ ID NO: 20), EFM = Enterococcus faecium (SEQ ID NO: 21), EFS = Enterococcus fococcus facicos cc facioc LB = Lactobacillus brevis (SEQ ID NO: 23), SM = S. Mutans (SEQ ID NO: 24), SG = Streptococcus gallolyticus (SEQ ID NO: 25), SUB = Streptococcus uberis (SEQ ID NO: 25) No. 26), SSU = Streptococcus p1 / 7 (SEQ ID NO: 27). It is a figure which shows the folic acid metabolism in a swine streptococcus. It is a schematic diagram of the estimated folate metabolism of Streptococcus pneumoniae. It is a graph which shows the expression level of orf2 and folC in a Streptococcus pneumoniae wild type isolate and a mutant. Expression levels of orf2 and folC (Panel A) in Streptococcus pneumoniae wild-type isolates strain 10 (black bar graph) and S735 (white bar graph) grown exponentially in Todd Hewitt medium, and Todd Hewitt medium Expression levels of orf2 and folC in strain S735 (panel) supplemented with empty control plasmids pCOM1 (black bar graph), orf2 [10] (white bar graph) or orf2 [S735] (hatched bar graph) grown exponentially in B). Culture in Todd Hewitt medium until early log phase (EEP) (white bar graph), log phase (EP) (thin diagonal bar graph), late log phase (LEP) (coarse diagonal bar graph) and stationary phase (SP) (black bar graph) Later, the expression level (panel C) of orf2 in S735 supplemented with orf2 [10], orf2 [S735] and orf2 [S735] [t488a] is shown. Expression levels were quantified using qPCR and presented as relative expression values for the housekeeping gene recA. Experiments were performed in triplicate. Error bars indicate the standard error of the mean value. Significance was determined by paired t test. ^* p <0.05, ^** p <0.01. It is a figure which shows the prediction three-dimensional structure of FolT protein of Streptococcus pneumoniae. It is a graph which shows the body temperature of the piglet after the Streptococcus pneumoniae infection of Experiment 1. The mean body temperature of piglets (n = 5) infected with either wild type strain 10 (pink) or strain 10ΔfolT (CBS140425) is shown. Error bars indicate the standard error of the mean value. It is a graph which shows the bacteremia value of the piglet after the infection with Streptococcus pneumonia in Experiment 1. Shown is the mean bacterial blood value of piglets (n = 5) infected with either wild-type strain 10 (pink) or strain 10ΔfolT (CBS140425) (blue). Error bars indicate the standard error of the mean value. It is a graph which shows the survival curve of the piglet infected with the Streptococcus pneumoniae of Experiment 1. Pigs were infected with either wild-type strain 10 or strain 10ΔfolT (CBS140425). Pigs were euthanized when they reached a predetermined humane endpoint for ethical reasons. Statistical analysis was performed using a log-rank (Mantel-Cox) test. It is a graph which shows the bacteriological examination of the piglet infected with the Streptococcus pneumoniae of Experiment 1. Pigs were infected with either wild-type strain 10 or strain 10ΔfolT (CBS140425). Bacteria were enumerated by serial dilution and plating. The number of bacteria was calculated as CFU / ml. Different colors indicated different piglets. It is a graph which shows the survival curve of the piglet infected with the Streptococcus pneumoniae of Experiment 2. Piglets (n = 10) were infected with either wild-type strain 10 or strain 10ΔfolT (CBS140425). Pigs were euthanized when they reached a predetermined humane endpoint for ethical reasons. It is a graph which shows the body temperature of the piglet after the Streptococcus pneumoniae infection of Experiment 2. The mean body temperature of piglets (n = 10) infected with either wild type strain 10 (blue) or strain 10 ΔfolT (CBS140425) (green) is shown. It is a graph which shows the walking movement of the piglet after the infection with Streptococcus pneumoniae of Experiment 2. The percentage of positive observation results in piglets infected with either wild-type strain 10 (blue) or strain 10ΔfolT (CBS140425) is shown. Severity of walking movement 1: Mild lameness 2: Moderate lameness or no attempt to stand up 3: Severe lameness (handled as a humane endpoint) It is a graph which shows the consciousness state of the piglet after the Streptococcus pneumoniae infection of Experiment 2. It is a graph which shows the ratio of the positive observation result in the piglet infected with either wild type strain 10 (blue) or strain 10 (DELTA) folT (CBS140425). Consciousness Severity 1: Depression, 2: Apathy, 3: Coma. FIG. 6 is a graph showing protection after vaccination of pigs with ΔFolT2 strain (CBS143192) and exposure to Streptococcus serotype 2 pigs. On days 1 and 21, pigs were vaccinated with ΔFolT2 strain (CBS143192). On day 35, approximately 2 × 10 ⁹ CFU of toxic Streptococcus serotype 2 isolates were exposed to animals by intraperitoneal (ip) administration. Animals were observed for signs of disease associated with Streptococcus pyogenes for 7 days after exposure. Necropsy was performed on animals that were dead or needed to be euthanized prior to off-test for animal welfare. Numbers indicate the percentage of animals that were lethal or euthanized after exposure (lethal rate). FIG. 6 is a graph showing protection after vaccination of pigs with ΔFolT strain (CSB140425) and exposure to Streptococcus pyogenes serotype 2. On days 1 and 21, pigs were vaccinated with ΔFolT strain (CBS140425). On day 36, approximately 2 × 10 ⁹ CFU of toxic Streptococcus serotype 2 isolates were exposed to the animals by intraperitoneal administration. Animals were observed for signs of disease associated with Streptococcus pyogenes for 7 days after exposure. Necropsy was performed on animals that were dead or needed to be euthanized prior to off-test for animal welfare. Numbers indicate the percentage of animals that were lethal or euthanized after exposure (lethal rate).

Introduction So far, the inventors have used complementation testing methods to identify novel virulence factors that may act as vaccine candidates. This test method is used to cause severe toxic shock-like syndrome in post-infection piglets and to death within 24 hours after infection ^[14] , a highly virulent porcine streptococcal isolate (S735-pCOM1- V [10]) was generated. After transformation with plasmid DNA isolated from approximately 30,000 stored clones with randomly cloned genomic DNA fragments from a toxic serotype 2 isolate (strain 10), S735-pCOM1-V [10] was selected from a library of clones generated with an attenuated serotype 2 isolate (S735). An isolated strain with increased toxicity was selected by infecting piglets with strain S735 containing a plasmid library of genomic fragments derived from strain 10. One epidemic clone isolated from an infected piglet contained a 3 kb genomic fragment from strain 10 named V [10] and was demonstrated to be highly toxic in subsequent animal experiments. V [10] contained an incomplete open reading frame (ORF) followed by two genes (orf2 and folC) in the operon structure and the second incomplete ORF. Assuming that only the full-length ORF could contribute to the virulent toxicity of this isolate, the inventors further characterized the orf2-folc-operon. The first ORF in the operon could not be annotated and was named orf2 as the second ORF in the operon showing homology to the gene encoding polyphoryl polyglutamate synthase (FolC). This operon was present in all swine streptococcal serotypes, including the parental strain S735. The less toxic strain S735 contained orf2-folc and several single nucleotide polymorphisms (SNPs) in the non-coding region compared to strain 10. Both genes of the operon with increased toxicity may be putative virulence factors, in which case they can be putative vaccine candidates. Here we have 1) whether orf2-folC-operon strong toxicity is caused by orf2, or by folC, or both, and 2) single nucleotide polymorphisms in the promoter region of orf2-folC-operon. The effect on toxicity was examined.

Materials and Methods Bacterial strains and plasmids Streptococcus pyogenes are cultured in Todd-Hewitt medium (Oxoid, London, United Kingdom) on a Columbia blood base agar plate (Oxoid) containing 6% (vol / vol) horse blood. Sowing. E. coli was cultured in Luria Broth and seeded on Luria Broth containing 1.5% (wt / vol) agar. If necessary, 1 [mu] g ml ^-1 erythromycin in Streptococcus suis, it was added 200 [mu] g ml ^-1 E. coli. The Streptococcus pyogenes strain S735 supplemented with a plasmid containing 3 kb of the genomic fragment derived from strain 10 (S735-pCOM1-V [10]) and other Streptococcus used in this study have been described so far. ^[14] (Figure 1).

(Example 1) Complementation test of Streptococcus pyogenes S735 Of two ORFs in the V [10] operon (ie, orf2 [10] or foC [10]) preceded by a putative promoter region of the operon from strain 10 S735 was supplemented with plasmid pCOM1 containing one of the two or plasmid pCOM1 containing the cognate upstream promoter from orf2 and S735 (orf2 [S735]) (FIG. 1). In order to construct such a plasmid, primers having restriction sites are orf2 [10] or orf2 [S735] (comE1-comE2), foC [10] (comE4-comE6) or the operon promoter region (comE1-comE3). Were designed to amplify (Table 1). The resulting PCR products orf2 [10] and orf2 [S735] were digested using the restriction enzymes SacI and BamHI, cloned into pKUN19 ^[15] , digested with the same restriction enzymes, and subsequently cloned into pCOM1, PCOM1-orf2 [10] and pCOM1-orf2 [S735] were obtained, respectively. The folC [10] PCR amplicon was digested using the restriction enzymes SmaI and BamHI and cloned into pKUN19 which was cleaved with the same restriction enzymes. A PCR product containing the promoter region of V [10] was cloned in front of folC [10] using restriction enzymes SacI and SmaI. Subsequently, the complete fragment of promoter V [10] -folc [10] was digested from pKUN19 using SacI and BamHI, cloned into pCOM1 cleaved with the same restriction enzymes, and pCOM1-folc [10] was cloned. Obtained. In vitro transcription / translation was performed using ³⁵ S-methionine to confirm that the fusion product of promoter-folC [10] was transcribed. A clear band of FolC molecular weight (46.8 kDa) was detected, demonstrating that the fusion product can be expressed and translated (FIG. 2). All plasmids were introduced into porcine Streptococcus strain S735 by electroporation. Furthermore, pCOM1-V [10] was introduced into non-toxic serotype 2 strain T15 by electroporation to obtain T15-pCOM1-V [10].

(Example 2) Experimental infection with complementary isolates Experimental infection of sterile piglets born by caesarean section was performed as previously described ^[14] . Prior to infection, the aseptic condition of the piglets was confirmed by inoculating swollen tonsils on a Columbia agar plate containing 6% horse blood. Briefly, 4 or 1 week old sterile pigs were infected intravenously with 10 ⁶ colony forming units (CFU) of Streptococcus pneumoniae, followed by 40 mg kg ⁻¹ body weight erythromycin (erythromycin-stearate) (Abbott BV, Amstelveen, The Netherlands) was orally administered twice daily to maintain selective pressure against the Streptococcus pneumoniae isolate carrying the pCOM plasmid. Infected pigs were monitored twice daily for tonsillar swabs collected for clinical signs and bacteriological analysis. Pigs were euthanized if they showed clinical signs of arthritis, meningitis, or sepsis after infection with Streptococcus pneumoniae. CNS, serosa and joint tissue samples were collected during necropsy, homogenized, and bacterial cell counts were determined by seeding serial dilutions on Columbia agar plates containing 6% horse blood and 1 μg ml ^-1 erythromycin. Quantified. In order to be able to compare the results from the various animal experiments contained herein, a uniform scoring system for non-specific and specific symptoms was applied to all animal experiments. Nonspecific symptoms included anorexia and depression, which were scored at 0 (none), 0.5 (mild anorexia / depression) or 1 (severe anorexia / depression). Specific symptoms include symptoms of lameness, central nervous system (CNS) symptoms (such as circulatory movement or walking in a circle, posterior arch reflex, nystagmus, as these are all symptoms of sepsis or serositis Motility disorders) as well as reverse hair, dorsal dorsum (back vertebral bay) and tremor. Based on these observations, a clinical index was calculated by dividing the number of observations in which either specific or non-specific symptoms were seen by the total number of observations for this parameter. This indicates the percentage of observations in which either specific or non-specific symptoms were seen. A similar method was adopted for “Fever Index”. Fever was defined as body temperature> 40 ° C. “Mean lethal days” was used as a survival parameter. After reaching the humane endpoint (HEP), the animals were euthanized, but the time from inoculation to reaching HEP also means the severity of the infection. This is calculated by averaging the survival rate in days from inoculation to lethality.

  Animal experiments with strain CBS140425 were conducted at the facility of Wageningen UR, Lelystad, NL's Central Veterinary Institute (currently called Wageningen Bioventional Research (WBVR)), and Wageningen UR, Lelystad of the Wageningen UR, Lelystad Approved in accordance with Dutch regulations regarding experiments (# 809.47126.04 / 00/01 and # 8700.471126.04 / 01/01). Animal experiments with strains CBS 140425 and 143192 were also performed in accordance with US regulations regarding animal experiments.

  Statistical analysis was performed using the nonparametric Kruskal-Wallis test for group clinical indicators (fever index, specific and non-specific symptoms) due to the lack of uniformity of variance between groups. In subsequent analyses, Mann-Whitney U test was used to compare all groups in pairs to the control group (S735-pCOM1) for all three parameters. Differences were considered statistically significant at p <0.05. Calculations were performed using SPSS19 (IBM, New York, USA).

(Example 3) Experimental infection with strain 10ΔfolT (CBS140425), Experiment 1
Ten 4-week-old piglets were reared at the CVI animal facility as two groups of 5 animals. The piglets were free to get food and clean water. Animals given warm light and playground equipment were available throughout the experiment. Prior to the start of the experiment, piglet tonsil swabs were screened for colonization of Streptococcus serotype 2 by PCR. Only PCR negative piglets were included in the experiment. Ten days later, either 1.1 × 10 ⁶ CFU wild type strain 10 or 9.2 × 10 ⁵ CFU mutant 10ΔfolT were infected by intravenous administration from the jugular vein. Prior to infection, the basal body temperature of the piglets was monitored daily for a period of 3 days. EDTA blood was collected prior to infection to obtain a pre-infection plasma sample, as well as a basal level of white blood cell (WBC) count. Infected pigs were monitored for clinical signs three times a day at 8 pm, 3 am and 9 am. Non-specific symptoms include anorexia and depression, while specific symptoms include lameness, central nervous system (CNS) symptoms (circulating movement or walking in a circle, opistotonus) ), Motility disorders such as nystagmus), and reverse hair, dorsal dorsi (post-spondylosis) and tremors, all of which were symptoms of sepsis or serositis. Tonsils and fecal swabs were collected daily for bacteriological analysis. Blood was collected daily for bacteriological analysis, WBC count and plasma collection. Pigs were euthanized if they showed clinical signs of arthritis, meningitis, or sepsis after infection with Streptococcus pneumoniae. At necropsy, the viscera (kidney, liver, spleen, peritoneum and pericardium) were bacteriologically screened for Streptococcus pneumoniae by seeding on Columbia agar plates containing 6% horse blood. Organs that were macroscopically infected with Streptococcus pneumoniae, such as pyogenic arthritis, pericarditis or peritonitis, were seeded at serial dilutions to determine the bacterial load. Tissue samples of such organs were fixed in formalin for histological examination. The animal experiment was approved by the Ethical Committee of the Central Veterinary Institute of Wageningen UR, Lelystad, The Netherlands according to the Dutch Code for Animal Experiments (# 2014011).

(Example 4) Experimental infection with strain 10ΔfolT (CBS140425), Experiment 2
In the second experiment, about 3 weeks old piglets (commercial hybrids) were used. The piglets are not vaccinated against Streptococcus pneumoniae, are from the PRRSV negative group, have not been given any drug-contaminated feed, and at registration, the tonsils by PCR for Streptococcus pyogenes serotype 2 The gland swab was negative. Treatment groups (10 piglets each) were raised separately. Animals were inoculated by intravenous administration with either 3.48E + 07 CFU wild type strain 10 or 1.45E + 07 mutant strain 10ΔfolT. Animals were observed once daily for 7 days for clinical signs of Streptococcus pneumoniae-related disease (eg, increased body temperature, lameness, and behavioral changes). Any animal showing clinical signs that reached the humane endpoint (eg, CNS signs, debilitating lameness) was euthanized to minimize pain. Euthanized animals were necropsied to identify lesions typically associated with swine streptococcal disease. Animals that survived until the end of the observation period were similarly euthanized and necropsied.

Example 5a: Vaccination of pigs with ΔFolT2 strain (CBS143192) and protection after exposure to swine streptococci serotype 2 Tests were carried out in commercial mating pigs. The pigs were 21 ± 7 days of age as of the first vaccination date. The animal has never been vaccinated against Streptococcus pneumoniae, is negative for tonsils swabs by PCR for Streptococcus pneumoniae serotype 2, is negative for PRRSV by serological tests, and for Streptococcus pneumoniae serotype 2 Derived from a female pig that was negative for tonsils swabs by PCR. Table 6 lists the test groups, vaccination routes and doses, days of vaccination, and schedule and routes of exposure. The media used are listed in Table 7.
On day 34, blood and tonsil swabs were collected from all animals, then strict control animals were transferred to another area, while all other groups were mixed. On day 35, approximately 2 × 10 ⁹ CFU of toxic Streptococcus serotype 2 isolates were exposed to animals by intraperitoneal (ip) administration.

Animals were observed for signs of disease associated with Streptococcus pneumoniae for 7 days after exposure. Necropsy was performed on animals that had been killed or euthanized prior to off-test for animal welfare. During necropsy, animals were evaluated for gross signs typically associated with swine streptococcal disease, and CNS (ie brain) and joint swabs were collected. In the off-test, all remaining animals were euthanized, necropsied and samples taken.
The preparation of vaccines and placebo is described in Table 7.
The preparation of the exposed substance is described in Table 8.
Vaccination with the Streptococcus pneumoniae ΔFolT mutant reduced the number of dead animals or animals that had to be euthanized for animal welfare during the post-exposure observation period (see Table 9 and FIG. 16). In addition, ΔFolT vaccination shows that the animal exhibits severe claudication findings (ie, the number of animals that cannot stand or do not attempt to stand up), as well as conditions that are very limited to being indifferent to the surroundings. (See Table 10 and Table 11).
During necropsy, signs of brain inflammation indicated by the presence of fibrin and / or cerebrospinal fluid were seen less frequently in ΔFolT vaccinated animals compared to negative controls (see Table 12).
From brain and joint swabs collected at autopsy from animals vaccinated with ΔFolT strain, Streptococcus pyogenes isolated isolates were recovered less frequently than negative controls (see Table 13 and Table 14).

Example 5b: Vaccination of pigs with strain 10ΔfolT (CBS140425) and protection after exposure to swine streptococcus serotype 2 Tested in 21 +/- 5 day old commercial mating pigs as of the first vaccination date Went. The animal has never been vaccinated against Streptococcus pneumoniae, is negative for tonsils swabs by PCR for Streptococcus pneumoniae serotype 2, is negative for PRRSV by serological tests, and for Streptococcus pneumoniae serotype 2 Derived from a female pig that was negative for tonsils swabs by PCR. The test groups, the number of animals / group at the start of the study, the vaccination dose, the number of days of vaccination, the vaccination route, the date of exposure and the route of exposure are listed in Table 15.
On day 35, blood and tonsil swabs were collected from all animals and strict control animals were euthanized. On day 36, approximately 2 × 10 ⁹ CFU of toxic Streptococcus serotype 2 isolates were exposed to the animals by intraperitoneal administration.

Animals were observed for signs of disease associated with Streptococcus pyogenes for 7 days after exposure. Necropsy was performed on animals that were dead or needed to be euthanized prior to off-test for animal welfare. During necropsy, animals were evaluated for macroscopic signs typically associated with swine streptococcal disease and CNS swabs were collected. In the off-test, all remaining animals were euthanized, necropsied and samples taken.
The preparation of vaccines and placebo is described in Table 16. The exposure material preparation is described in Table 17.
With the Streptococcus pyogenes FolT mutant, the number of animals exhibiting lameness, the number of animals exhibiting abnormal behavior (ie depression, coma) after exposure and the need to be euthanized for animal welfare during the lethal animal or post-exposure observation period The number of animals that were present decreased (see Table 18, Table 19 and Table 20 and FIG. 17).
Off-test (ie, 7 days after exposure or release from study due to lethality or euthanasia), abnormal findings in the brain (ie fibrin, cerebrospinal fluid) and abnormal findings in the thoracic cavity (ie fibrin, pleural effusion, pulmonary congestion, pneumonia) Animals were observed for. In addition, a sample was taken from the brain for recovery of Streptococcus pneumoniae. The results are listed in Table 21, Table 22 and Table 23.

Example 6: cDNA synthesis and quantitative PCR
RT-PCR
Using 200 ng of RNA, 25 ng μl ⁻¹ random primers (Promega, Madison, WI, USA), 10 mM dNTP (Promega), 10 mM DTT (Invitrogen), 40 U RNAsin (Promega) and CDNA was synthesized according to the manufacturer's instructions in a reaction using SuperScript II reverse transcriptase (Invitrogen).
qPCR
The cDNA was diluted 20 times for qPCR analysis. Primers were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA) (Table 1). In each reaction, 12.5 pmol forward primer, 12.5 pmol reverse primer and POWER SYBR Green PCR Master Mix (Applied Biosystems) were used according to the manufacturer's instructions. QPCR was performed using ABI7500 (Applied Biosystems). The most stably expressed reference gene was quantified using GeNorm software (GeNorm). In Streptococcus pneumoniae, the variability of recA is highest in the expression of the six potential reference genes tested (phosphoglycerate acid dehydrogenase (pgd), acetyl CoA acetyltransferase (aca), mutS, glutamate dehydrogenase (gdh)). There were few. Genorm integrates the expression data into a numerical value indicating the stability of expression, where 1 indicates the most stable gene. The stable number of Streptococcus pneumoniae ranged from 1.667 for gdh to 1.217 for recA. The level of expression of such a reference gene was measured to adjust for differences in RNA recovery conditions and RT reaction conditions. In each qPCR run, a calibration curve consisting of vector data containing the cloned PCR product of the target gene in the reaction was incorporated. A standard curve was constructed from data for seven 10-fold dilutions of the control vector. Thus, both the expression level of the target gene and the expression level of the external reference gene can be calculated from the calibration curve. In each reaction, water was used to place the cDNA or template as a negative control. Analysis was performed using ABI7500 software (Applied Biosystems).

Sequence analysis Sequencing reactions and sequence analysis were performed by Baseclear (Leiden, The Netherlands).

(Example 7) Site-directed mutagenesis Site-directed mutagenesis was achieved using a Quick-change II site-directed mutagenesis kit (Agilent Technologies, La Jolla, CA, USA) according to the manufacturer's instructions. PCR primers were designed by the attached software (manufactured by Agilent Technologies) (Table 1). The plasmid pCOM-orf2 [S735] was amplified by using primers t448a and t488a_antisense, and the -35 region of the putative promoter region of the orf2-folc-operon of S735 was changed from 5'-TGGTCA-3 'to 5' -A desired mutation was introduced that changes up to TGGACA-3 '(Figure 1). The reaction mixture was digested using DpnI to inactivate the original template vector and subsequently transformed into XL-1-blue competent cells (Invitrogen). In order to exclude the possibility of PCR errors in the vector backbone, the plasmid insert (orf2 [S735]) was isolated from the template vector after digestion with the restriction enzymes BamHI and SacI and cloned into pCOM1 which was degraded by the restriction enzymes. Turned into. The resulting plasmid was introduced into Streptococcus pneumoniae isolate S735 by electroporation, transformants were selected on Columbia agar containing ¹ μg ml ⁻¹ erythromycin, and S735-pCOM1-orf2 [S735] [t488a ] Was obtained. Sequencing was used to rule out the presence of PCR errors in the final construct.

Example 8 Construction of Streptococcus folate Substrate Binding Protein (folT) Knockout Mutant A syngeneic folT knockout mutant was constructed in strain 10 by destroying folT using a spectinomycin resistance cassette. pCOM1-V [10] ^[14] was digested with BamHI and ligated to pKUN plasmid digested with BamHI to obtain pKUN-V [10]. In order to remove the 3 ′ portion of V [10], pKUN-V [10] was digested with SphI, and then the vector fragment was purified and ligated to obtain pKUN-V [10] ^* . pKUN-V [10] ^* was partially digested with XmnI, and the linear vector fragment was purified and ligated with a blunt-end spectinomycin resistance cassette to obtain pKUN-V [10] ^* -Spec ^R. For construction of the mutant strain, V [10] ^* -Spec ^R was amplified by PCR using V735-fw and M13-rev. The PCR product was purified using PCR Purification Kit (Qiagen). The purified PCR product was used to transform Streptococcus pneumoniae strain 10 using ComS as a ^[16] competence inducer as described by Zaccaria et al. To induce homologous recombination. Transformants were selected on Columbia agar plates containing 6% (vol / vol) horse blood and 100 μg ml ⁻¹ spectinomycin. Double crossovers were checked by PCR and confirmed using Southern blotting. To exclude the possibility of point mutations, the chromosomal DNA of a syngeneic knockout mutant was isolated and transformed into strain 10. Further, mutant strains were selected on a Columbia agar plate containing 6% (vol / vol) horse blood and 100 μg ml ⁻¹ spectinomycin and screened by PCR to obtain strain 10ΔfolT. This prototype recombinant ΔFolT mutant strain was deposited as “CBS140425 Streptococcus pneumoniae ΔFolT mutant strain” at the Centralalburau room Scheme cultures on August 19, 2015 for the purpose of patent procedures based on the laws of the Budapest Treaty.

Example 9 ΔfolT deletion mutant not containing spectinomycin resistance gene A ΔfolT deletion mutant not containing spectinomycin resistance gene was also constructed. For this purpose, a temperature sensitive shuttle vector pSET5s (Takamatsu D., Osaki, M. and Sekizaki, T. 2001. Plasmids 46: 140-148) was used. Plasmid pSET5s contains a temperature sensitive origin of replication and can be grown at 37 ° C. in E. coli, but plasmid replication is blocked above 37 ° C. in Streptococcus pneumoniae (Takamatsu et al). pSET5s contains a chloramphenicol resistance gene (Cm), which can be used for selection of transformants in E. coli as well as Streptococcus pneumoniae. The prototype recombinant ΔFolT mutant not containing the spectinomycin resistance gene was designated as “CBS143192 swine Streptococcus ΔFolT2 mutant” at Westerijk Fungal Biodiversity Institute for the purpose of patent procedure based on the laws of the Budapest Treaty on August 25, 2017. Deposited on the day.

  In order to construct a ΔfolT mutant isolate, PCR products containing 5'- and 3'-flanking sequences of the foT gene were generated. This fragment is cloned into pSET5s and Cm resistant transformants are selected at 37 ° C. in E. coli. The plasmid was then isolated from E. coli and introduced into swine streptococcus strain 10. Transformants were selected on a 30 ° C. Columbia agar plate containing Cm. The transformed colonies were used to inoculate 1 ml of Todd Hewitt medium (THB) containing Cm and the culture was grown overnight at 30 ° C. This overnight grown culture is diluted 100-fold with the same medium and incubated as above until the optical density at 600 nm reaches 0.2-0.3, where the culture reaches 38 ° C. . At this temperature, the plasmid cannot replicate. By this step, a strain in which the plasmid is integrated into the chromosome by a single recombination phenomenon is selected. Serial dilutions of this culture were seeded on Columbia horse blood plates containing Cm. Plates were incubated overnight at 38 ° C. Colonies containing the recombinant plasmid integrated into the chromosome were selected, seeded in 1 ml of Todd Hewitt medium (THB) containing Cm, and incubated at 38 ° C. overnight. Cultures were diluted 100-fold with THB without Cm and grown at 28 ° C. for passage after 5 passages. At this temperature, the plasmid can replicate and is excised from the chromosome by a second recombination event against the replicated target gene sequence. Excision of the plasmid can give rise to a wild type genotype or a folT deletion mutant. Serial dilutions of the cultures were seeded on Columbia horse blood plates (without Cm) and incubated overnight at 38 ° C. Single colonies were then replica plated on Columbia horse blood plates with or without Cm. Cm sensitive colonies were screened by PCR to identify ΔfolT mutant isolates that did not contain the spectinomycin resistance gene.

Hybridization test Chromosomal DNA was isolated from a stationary culture of Streptococcus pyogenes. 200 nanograms of purified DNA was marked on Genescreen-Plus (Perkin Elmer, USA). Labeling of the probe with ³² P, hybridization and washing were performed as previously described ^[17] . The folT and folC PCR products were used as probes, while the 16S rRNA probe was used as a positive control.

Overexpression of folT sufficient to induce strong toxicity in S735 strain The introduction of V [10], a 3 kb genomic fragment derived from toxic serotype 2 strain 10, increased the toxicity of attenuated serotype 2 strain S735. ^[14] , a highly toxic isolate (S735-pCOM1-V [10]) was produced. All pigs infected with S735-pCOM1-V [10] died within 1 day after infection (pi), while pigs showed a high proportion of severe clinical signs (Table 2) On the other hand, almost all pigs infected with the control strain S735-pCOM1 survived throughout the experiment. Clinical indicators were significantly different between S735-pCOM1-V [10] and pigs infected with S735-pCOM1 (p ≦ 0.01) (Table 2). As a control, we also tested the toxicity of S735 transformed with a plasmid containing a homologous 3 kb fragment from S735 (S735-pCOM1-V [S735]). Pigs infected with S735-pCOM1-V [S735] survived a high rate throughout the experiment. In contrast, pigs infected with S735-pCOM1-V [S735] showed significantly more specific clinical signs than pigs infected with S735-pCOM1 (p ≦ 0.01) (Table 2), Differences in clinical indicators of fever and nonspecific symptoms were not significantly different between groups (p = 0.06). Thus, the increase in the copy number of V [S735] in S735 by introduction of plasmid pCOM1-V [S735] increased the specific clinical signs of Streptococcus pneumoniae. Nevertheless, specific and non-specific clinical signs resulting from swine infection with S735-pCOM1-V [10] are significantly increased compared to pigs infected with S735-pCOM1-V [S735]. (P ≦ 0.01), it was demonstrated that introduction of V [10] in strain S735 increased toxicity over introduction of V [S735]. This result indicated that the strong toxicity of strain S735pCOM-1-V [10] can be attributed to various base polymorphisms in V [10] compared to V [S735].

To determine whether both the orf2 and folC genes are required for the observed increase in toxicity, both genes for the operon from strain 10 preceded by the cognate promoter sequence were introduced separately into strain S735. Strains S735-pCOM1-orf2 [10] and S735-pCOM1-folc [10] were produced. The toxicity of such isolates was determined in experimental infections of piglets using S735-pCOM1-V [10] and S735-pCOM1 as controls. Table 2 shows that pigs infected with S735-pCOM1-V [10] or S735-pCOM1-orf2 [10] died within one day after infection with severe clinical signs. Infected pigs developed a toxic shock-like syndrome that was not seen when using wild-type strain 10 in an experimental infection, and fragments V [10] and orf2 [10] increased the toxicity of S735, resulting in strain 10 More toxic isolates were obtained ^[3] . Both specific and non-specific symptoms were significantly increased in pigs infected with S735-pCOM1-V [10] or S735-pCOM1-orf2 [10] compared to S735-pCOM1 (p <0.01). (Table 2).

Bacteriological examination showed that Streptococcus pneumoniae colonized with high CFU in the CNS, serosa and joints. In contrast, pigs infected with S735-pCOM1-folC [10] or S735-pCOM1 survived throughout the experiment (11 days after infection) and showed mild infectious symptoms such as fever. There was no significant difference in clinical results between A735-pCOM1-folC [10] and pigs infected with S735-pCOM1. This clearly demonstrated that the introduction of folC [10] does not increase the toxicity of strain S735, but the introduction of V [10] and orf2 [10] increases the toxicity of strain S735. The inventors therefore concluded that the increased toxicity of S735-pCOM1-V [10] observed compared to S735-pCOM1 is due to the introduction of orf2 [10].
In conclusion, both the copy number of V [10] and the genetic background of the orf2-folC operon are believed to be determinants of toxicity for a given isolate.

Measurement of in silico data demonstrates that ORF2 is a substrate that binds to a protein that promotes folate transport. The increased toxicity was attributed to the introduction of multiple copies of orf2 [10], so we investigated the putative function of orf2. . In silico analysis of the 5 ′ intergenic region preceding the orf2-foC operon revealed the presence of the predicted secondary structure, which showed strong homology to the tetrahydrofolate riboswitch (FIG. 3). Tetrahydrofolate riboswitches are a class of homologous RNAs in certain bacteria that bind to tetrahydrofolate (THF) ^[18] . It is located almost exclusively in the putative 5 'untranslated region of protein-coding genes, and many of these genes are known to encode either folate transporters or enzymes involved in folate metabolism. ing. For these reasons, it was speculated that RNA functions as a riboswitch. THF riboswitches have been found in various pharmacutes, in particular Clostridiales and Lactobacilles, and more rarely in other bacterial strains. The THF riboswitch was one of many conserved RNA structures found in projects based on comparative genomics ^[19] . A relationship with folate metabolism was confirmed by Eudes et al. And it was demonstrated that the gene located upstream of folc in Streptococcus pneumoniae encodes the folate transporter FolT ^[20] . This annotation was also applied to the novel genomic sequence SC0707071 of Streptococcus pyogenes, where it was annotated that the homologous gene of ssu0135 encodes FolT (GenBank AGG633648.1). The three-dimensional structure of Lactobacillus brevis folate energy-coupled factor transport was established ^[21] , and a multiple protein model of the folate transporter was proposed. In this model, ORF2 / FolT functions as a transmembrane substrate-specific binding protein. Together with the more general transmembrane protein and two nucleotide binding proteins that form an energy coupling module, this complex facilitates transmembrane transport of folic acid. Only substrate binding protein (FolT) is specific for folic acid and other elements are also used for transport of other substrates such as thiamine or riboflavin. When comparing the FolT protein sequence of Streptococcus pneumoniae with the putative FolT sequence of other organisms, it is clear that conserved amino acids are also conserved in Streptococcus pneumoniae ^[21] (Figure 4). Interestingly, FIG. 4 also shows that arginine was very well conserved in the human folate transporter as well as in E. coli. In FIG. 4, red indicates small hydrophobic amino acids (including aromatic-Tyr), blue indicates acidic amino acids, magenta indicates basic amino acids, and green indicates hydroxyl, sulfhydryl amines and Gly. The tetracycline transporter is also conserved in Streptococcus pneumoniae (Arg99) folT, suggesting that this residue is important for transporters ranging from human to bacterial ^[22] . Taken together, such data strongly suggests that ORF2, a conserved protein of unknown function, identified using a complementation test, encodes a substrate binding protein that promotes folate transport.

All enzymes required for the synthesis of tetrahydrofolate (THF) via the classical folate biosynthetic pathway by sequence analysis of folate transport P1 / 7 (highly homologous to the genome of strain 10) Is encoded by Streptococcus pneumoniae (FIG. 5). Based on the data available in the KEGG database (www.kegg.jp), folic acid metabolism in Streptococcus pneumoniae is shown in the scheme as follows: folE, folQ, folB, folK, folC, folA and the substrates GTP, p-aminobenzoic acid The classical folate pathway is utilized using acid (PABA) and glutamyl (GLU). However, due to the additional genes present on the V [10] operon, Streptococcus pyogenes can also induce the expression of folT to directly transfer folic acid and use co-inducible expression of additional copies of folC. It is believed that folic acid can be immediately processed into the final product, tetrahydrofolic acid (THF). In this way, the combination of folT and folC forms an additional “shortcut”, which allows the generation of THF. The presence of a THF riboswitch located upstream of the folT-folC operon suggests tight regulation of this two-gene operon, which is expressed only under specific conditions, eg, folate-poor conditions. Can mean that. Based on the results of the above animal experiments, it is suggested that the folT-folc operon is expressed at least under in vivo conditions.

Presence and expression of folT in Streptococcus pyogenes The presence of the folT gene was demonstrated in all swine streptococci serotypes except serotype 32 and serotype 34. However, serotype 32 and serotype 34 were reassigned to belong to the genus Streptococcus orisratti instead of Streptococcus pneumoniae ^[1] . Thus, in conclusion, all Streptococcus serotypes are considered to have genes encoding FolT and FolC.

Sequence analysis of the orf2 putative promoter revealed a nucleotide position difference in the -35 region (TGGACA) of the putative promoter in strain 10 compared to strain S735 (TGGTCA) ^[14] . The effect of this SNP on the expression levels of orf2 and folC in strains 10 and S735 was determined using qPCR analysis. Significantly higher levels of orf2 and folC expression were observed in strain 10 compared to strain S735 (FIG. 6A). This clearly indicates that SNPs in the -35 region of the putative promoter affect orf2 and folc transcription. This demonstrates that the identified SNP is actually located in the promoter region that co-transcribes orf2 and foC in the operon. Furthermore, when pCOM1-orf2 [10] was introduced into S735, the expression of orf2 increased 31-fold compared to the introduction of pCOM1, but when pCOM1-orf2 [S735] was introduced, the expression of orf2 increased by a factor of five. (FIG. 6B). As expected, the expression level of folC was similar in both recombinant strains (FIG. 6B). To confirm that the identified SNP in the -35 region of the promoter was responsible for the transcriptional differences of orf2 in strains S735 and 10, when it was found in the promoter of orf2 [10], orf2 [S735] TGGTCA was mutated to TGGACA (strain S735-pCOM1-orf2 [S735] [t488a] was obtained). The transcription level of orf2 in S735-pCOM1-orf2 [S735] [t488a] is similar to the transcription level of strain S735-pCOM1-orf2 [10], and transcription of strain S735-pCOM1-orf2 [S735] in various growth phases It was shown to be 4 times higher than the level (FIG. 6C). Both promoters are most active early in the growth phase of Streptococcus pyogenes when cultured in Todd Hewitt medium (FIG. 6C). Taken together, these results indicate that in strain 10, the promoter located upstream of the orf2-folC-operon is stronger than the promoter located upstream of this operon in strain S735 because of the SNP in the -35 region. Demonstrate clearly that there is.

To determine whether SNPs associated with increased expression of the orf2-folc operon were associated with specific clonal or serotypes of Streptococcus pneumoniae, the promoter regions of multiple collections of isolates were sequenced. (Table 3). All isolates used were recently characterized and classified by CGH and MLST ^[23] . Based on the sequence data obtained, isolates can be classified into two main groups (Table 3). A strong -35 promoter region was found exclusively in serotype 1 and 2 isolates, which belonged to CGH cluster A and MLST clone complex 1 and expressed EF protein. SNPs with low promoter activity are of serotype 7 and 9 isolates belonging to CGH group B that are all negative for EF expression (except for two) and of large structure EF protein (EF ^* ) It was found in attenuated isolates of serotype 2 belonging to CGH group A / clone complex 1 (CC1) that are positive for expression. There are two exceptions here. Serotype 7 isolate (C126) that belongs to CC1, but does not express an EF protein containing a SNP associated with a stronger promoter, and a serum with a different -35 promoter sequence (TTGTCA) with undefined promoter strength Type 7 isolate (7711). In conclusion, only CC1 isolates (and one serotype 7 isolate) expressing EF protein contain SNPs associated with strong promoter activity. Because isolates of this combination of phenotype and genotype strongly correlate with toxicity ^[23 , ^24] , we have found that a strong promoter located upstream of the orf2-folc-operon It can be concluded that it is associated with a toxic isolate. This finding indicates that with the increase in toxicity observed after the introduction of additional copies of folT [10], increasing the expression of folT, either by increasing copy number or by a stronger promoter, increases the toxicity of Streptococcus pyogenes. I suggest that.

Growth of Streptococcus pneumoniae with an additional copy of folT in culture or without folT In culture of Streptococcus pneumoniae with an additional copy of folT or without functional folT, In comparison, there was no significant difference in proliferation.

Protein expression of FolT Based on the protein sequence of FolT, it was predicted that FolT is a very hydrophobic protein with at least 5 transmembrane domains. FolT 3D structure of Streptococcus pyogenes was predicted by homologous modeling (Expacy server) using 6 known FolT structures, among them the published 3D structure of FolT from Lactobacillus brevis (Figure 7).

FolT is important for survival in vivo: Toxicity of folT knockout strain 10ΔfolT Because folT is strongly increased by overexpression of folT in attenuated porcine Streptococcus strains, we have found that FolT plays an important role in vivo Assuming that To test whether this assumption is correct, a syngeneic knockout was constructed in virulent swine streptococcus strain 10 by inserting a spectinomycin resistance cassette into the folT gene. Because folT and folC are in the operon structure, this knockout can also cause an additional copy of folC to be knocked out. To determine if folate transport is essential for toxicity in vivo, in Experiment 1, 10 pigs were infected intravenously with either wild type strain 10 or knockout strain 10ΔfolT. All pigs responded to this sowing and body temperature increased (Figure 8). However, pigs infected with wild-type strain 10 showed higher body temperatures than longer periods compared to pigs infected with knockout strain 10ΔfolT. This was also reflected by the difference in the fever index (the percentage of observations that pigs showed fever) between the two groups (p = 0.06). This suggests that the strain 10ΔfolT is less purulent compared to the wild type strain. This may be the result of the fact that significantly fewer bacteria were isolated from the blood of piglets infected with strain 10ΔfolT. Only 2 pigs infected with strain 10ΔfolT show a shorter bacteremia period compared to 5 pigs infected with strain 10, and pigs infected with strain 10 also have a bacterial count in these blood. It was significantly higher for longer periods (Figure 9). This suggests that strain 10ΔfolT is more efficiently removed from the blood or cannot survive in the blood. The white blood cell count revealed that pigs infected with wild type strain 10 showed a strong increase in WBC for a longer period. All pigs infected with strain 10 showed an increase in WBC, but only one of the pigs infected with strain 10ΔfolT showed an increase in WBC. The calculated WBC index is significantly different between groups (Table 5). Survival rates between the two groups were significantly different, pigs infected with strain 10 had an average survival of 2.6 days after infection, while pigs infected with strain 10 Δfolt survived 6.2 days after infection (FIG. 10). Pigs were euthanized when a predetermined humane endpoint was reached, but survival reflects the severity of the infection. As shown in FIG. 10, survival curves are significantly different between groups. Macroscopically, 4/5 of pigs infected with strain 10 showed specific clinical signs for infection with swine streptococcal strains such as arthritis, pleurisy, pericarditis or peritonitis, but strain 10ΔfolT It was revealed that 3/5 of the infected pigs showed specific clinical signs. Bacteriological examination of all infected organs revealed that the wild-type strain 10 was colonized with a higher bacterial load in more organs than the strain 10ΔfolT (FIG. 11).

The second animal experiment (Experiment 2) generally confirmed the data obtained in Experiment 1. As in the first experiment, the survival curves of wild-type strain 10 and strain 10ΔfolT isolates were significantly different. In experiment 2, all animals inoculated with strain 10ΔfolT survived until the end of the experiment, but 60% of animals inoculated with strain 10 had to be euthanized during the course of the experiment (FIG. 12). Furthermore, the frequency and severity of clinical signs (eg, body temperature, locomotor activity and consciousness, see FIGS. 13, 14 and 15) were significantly different between animals inoculated with wild-type strain 10 and strain 10ΔfolT. . The frequency of lesions due to gross findings in the joints and peritoneum obtained at necropsy also differed significantly between the wild type and 10ΔfolT mutant isolates.
Based on the results of infection experiments in piglets, it was concluded that the syngeneic knockout mutant 10ΔfolT was strongly attenuated compared to the wild type strain. This indicates that the folate transporter is required for bacterial survival under in vivo conditions. To summarize the results from both trials, such experiments showed that ΔfolT isolates resulted in near-zero mortality, minimal clinical signs, and reduced frequency of arthritis and peritonitis compared to the parental strain. Clearly shown. Therefore, it can be concluded that the ΔfolT strain is highly attenuated and safe.

Summary Results Vaccines containing bacteria (ΔfolT isolates) with modifications such as mutations, deletions or insertions in the DNA region encoding the bacterial folate substrate binding protein are exposed to unmodified bacterial toxic isolates. Defend the host from
Selecting a parent bacterial strain that is generally capable of folate transport and folate synthesis, and mutations, deletions or insertions in the DNA region encoding the bacterial folate substrate binding protein (known as the foT gene in Streptococcus pyogenes) Select bacteria from this parent strain for modification, and select bacteria with the ability to grow to a similar rate as the parent strain in vitro but to a lower rate compared to the parent strain in vivo A method of producing bacteria, preferably for use in a vaccine, preferably for use in a vaccine that provides protection from bacterial infection. Selecting a parent bacterial strain that is generally capable of folate transport and folate synthesis, and preferably by recombination means, in the DNA region encoding the bacterial folate substrate binding protein (known as the folT gene in Streptococcus pyogenes) , By transforming bacteria derived from this parent strain by modification such as deletion or insertion, and in vitro it grows to the same rate as the parent strain, but in vivo it has a lower rate compared to the parent strain And further selecting a bacterium having the ability to grow to, preferably for use in a vaccine, preferably for producing a bacterium for use in a vaccine that provides protection from bacterial infections. Such bacteria, as provided herein, still have the ability to produce folic acid for its own use by applying its novel folate synthesis pathway. If such a synthetic pathway is left intact, its ability to grow in vitro remains unaffected, but surprisingly, its growth and toxicity in the host (in vivo) is strongly reduced. Is shown.

Such bacterial strains that grow well in vitro but do not grow as well as their parent strain in vivo and are associated with a strong reduction in toxicity in vivo are very useful as vaccine strains. In such strains or mutants provided by the present invention, on the one hand, they are essentially unaffected in folic acid synthesis and are therefore capable of growing to high titers and are therefore relatively easy and inexpensive to produce, Therefore, since the growth in the host is reduced and the toxicity is reduced compared with the parent strain, such a strain or mutant is relatively harmless after in vivo application and is extremely useful as a vaccine against bacterial infections. Become.
In the first series of experiments herein, about 3 weeks old piglets (commercial hybrids) that have not been vaccinated against Streptococcus pneumoniae and have never received a drug-contaminated diet are available. Used for testing. The animals were tonsillar swab negative by PCR for porcine Streptococcus serotype 2 at the time of enrollment and were derived from the PRRSV negative group. Two treatment groups were housed separately in the test facility.

  Upon arrival at the study facility, blood and tonsil swabs were collected from all animals. After a suitable habituation period, on day 0 of the study, one group of animals was vaccinated with strain 10ΔFolT. Another group of animals was left unvaccinated. The same dose of mutant isolate was each revaccinated on the right side of the neck of the animal vaccinated on day 21. Animals were observed for local and systemic reactions after each vaccination. On day 35, blood and tonsil swabs were collected from all animals before exposing both groups of animals to the exposed strain ATCC 700794. Animals were observed for signs of Streptococcus pneumoniae-related disease (eg elevated body temperature, lameness, abnormal behavior, CNS signs) for 7 days after exposure. Necropsied animals were either dead or needed to be euthanized prior to off-test for animal welfare. During necropsy, animals were evaluated for gross signs typically associated with swine streptococcal disease (eg, CNS, joint, chest cavity inflammation). In addition, CNS swabs were collected for recovery of exposed isolates. On day 42, all remaining animals were euthanized, necropsied and samples were collected as described above. Vaccinated animals showed very little signs of streptococcal disease after exposure.

  A second series of experiments was performed in commercial mating pigs. The pigs were 21 ± 7 days of age as of the first vaccination date. The animal has not been vaccinated against Streptococcus pneumoniae and is Streptococcus serotype 2 and serologically it is PRRSV negative for tonsillar swab negative and Porcine Streptococcus serotype 2 for tonsils Derived from a female pig that was swab negative. Upon arrival at the study facility, blood and tonsil swabs were collected from all animals. After an appropriate acclimation period, on day 0 of the study, one group of animals was vaccinated with strain ΔFolT2 on the left side of the neck. Another group of animals was left unvaccinated. The same dose of mutant isolate was revaccinated on the right side of the neck of the animal vaccinated on day 21. Animals were observed for local and systemic reactions after each vaccination. On day 34, blood and tonsil swabs were collected from all animals, then strict control animals were transferred to another area, while all other groups were mixed. On day 35, toxic Streptococcus pneumoniae serotype 2 isolates were exposed to animals by intraperitoneal (ip) administration.

  Animals were observed for signs of disease associated with Streptococcus pyogenes for 7 days after exposure. Necropsy was performed on animals that were dead or needed to be euthanized prior to off-test for animal welfare. During necropsy, animals were evaluated for gross signs typically associated with swine streptococcal disease, and CNS (ie brain) and joint swabs were collected. In the off-test, all remaining animals were euthanized, necropsied and samples taken. Vaccination with the ΔfolT mutant reduced the number of dead animals or animals that had to be euthanized for animal welfare during the post-exposure observation period. During autopsy, signs of inflammation in the brain indicated by the presence of fibrin and / or cerebrospinal fluid were seen less frequently in ΔfolT2 vaccinated animals compared to negative controls. From brain and joint swabs collected at necropsy from animals vaccinated with the ΔfolT2 strain, swine streptococcal-exposed isolates were recovered less frequently compared to negative controls.

References

Table 1. Primer sequences

Table 2. Toxicity of complementary swine streptococci strains in sterile piglets; all strains contained a plasmid (pCOM1) with or without an insert. V [10] / V [S735]: strain 10 selected from library or original 3 kb fragment from strain S735; orf2 [10]: orf2 from V [10]; folC [10]: encodes dihydrofolate synthase Orf3 derived from V [10].
^a Proportion of pigs killed due to infection or needed to be sacrificed for animal welfare
^b Proportion of pigs with specific symptoms
^c Proportion of observations in experimental groups with specific symptoms (ataxia, lameness and / or rest of at least one joint)
^d Proportion of observations in experimental groups with nonspecific symptoms (anorexia and / or depression)
^e > Proportion of observations in experimental group with body temperature of 40 ° C
^fPast experiments (Smith et al., 2001) were reanalyzed to allow statistical comparisons between experiments. This reanalysis required new and rigorous definitions of specific and non-specific symptoms as described in Materials and Methods.
^* p ≤ 0.05 Compared to S735-pCOM1
^** p ≤ 0.01 Compared to S735-pCOM1
^g Serosa is defined as the peritoneum, pericardium or pleura.

Table 3. Sequence analysis of the -35 region of the orf2 / folC promoter between various Streptococcus isolates and serotype ^1
One Streptococcus isolate was described in de Greeff et al. [23].
² * indicates the high molecular weight form of MRP, and s indicates the low molecular weight form of MRP.
³ * indicates the high molecular weight form of EF.
^Four comparative genomic hybridizations (CGH) [23] were used to identify the genotype of all isolates.
⁵ This isolate belongs to clone complex 1.
⁶ Number of isolates analyzed / number of isolates with each -35 promoter sequence

Table 4. Clinical parameters of pigs infected with Streptococcus pyogenes, experiment 1
^* Compared with p ≦ 0.05 10
^** Compared with p ≤ 0.01 10
^# Compare with p ≤ 0.1 10

Table 5. Gross lesions showing arthritis and peritonitis:% positive observation in animals exposed to wild-type strain 10 and 10ΔFolT mutant isolate, experiment 2

Table 6. Test plan (for CBS143192 use test)

Table 7. Vaccine and placebo preparation (for CBS143192 use trial)

Table 8: Preparation of exposure (for CBS143192 use test)

Table 9. Percentage of animals killed or euthanized after exposure (lethal rate) (for use with CBS143192)

Table 10. Percentage of animals showing severe lameness after exposure (for use with CBS143192)

Table 11. Percentage of animals showing apathy after exposure (for use with CBS143192)

Table 12. Percentage of animals showing signs of brain inflammation at necropsy (for studies using CBS143192)

Table 13. Percentage of animals that recovered Streptococcus pneumoniae from brain swabs collected at necropsy (for tests using CBS143192)

Table 14. Proportion of animals that recovered Streptococcus pneumoniae from joint swabs collected at necropsy (for testing using CBS143192)

Table 15. Vaccination exposure test summary (for CBS140425 use test)

Table 16. Vaccine preparation (for CBS140425 use test)

Table 17. Preparation of exposure (for CBS140425 use test)

Table 18. Percentage of animals showing lameness after exposure (CBS140425)

Table 19: Percentage of animals with abnormal behavior after exposure (CBS140425)

Table 20: Percentage of animals killed or euthanized after exposure (lethal rate) (CBS140425)

Table 21: Proportion of animals with abnormal findings in the brain at necropsy (CBS140425)

Table 22: Proportion of animals with abnormal findings in the chest cavity at necropsy (CBS140425)

Table 23: Proportion of animals that recovered Streptococcus pneumoniae from brain swabs (CBS140425)

Claims (38)

  A ΔFolT mutant strain of bacteria having reduced ability to transport folic acid, wherein the ability is reduced by functionally deleting the folate transporter (FolT) function.   The ΔFolT mutant according to claim 1, which can be classified as streptococci, preferably as swine streptococci.   The ΔFolT mutant according to claim 1 or 2, which has an ability to synthesize folic acid.   The ΔFolT mutant according to any one of claims 1 to 3, wherein the expression of FolT is decreased.   5. A peptide domain FYRKP mutation or deletion, or a mutation or deletion in peptide domain FYRKP, or an insertion in peptide domain FYRKP, preferably having a mutation in R in peptide domain FYRKP. The ΔFolT mutant described in 1.   The ΔFolT mutant strain according to any one of claims 1 to 5, which was deposited as a “CBS140425 Streptococcus pneumoniae ΔFolT mutant strain” on 19th August 2015 at the Centralbureau room Scheme Cultures.   The ΔFolT mutant strain according to any one of claims 1 to 5, which was deposited on August 25, 2017 at the Westerijk Fungal Biodiversity Institute as a “CBS143192 Streptococcus pneumoniae ΔFolT2 mutant strain”.   The bacterial culture according to any one of claims 1 to 7.   A composition comprising the bacterium according to any one of claims 1 to 7 or the culture according to claim 8.   An immunogenic composition comprising the bacterium according to any one of claims 1 to 7 or the culture according to claim 8.   Use of the composition according to claim 9 or claim 10 for the production of a vaccine.   A vaccine comprising a bacterium according to any one of claims 1 to 7, a culture according to claim 8, or a composition according to claim 9 or claim 10. a. A dispenser for administering a vaccine to an animal, preferably a pig;
b. A ΔFolT mutant according to any one of claims 1 to 7, or a culture according to claim 8, a composition according to claim 9 or 10,
c. A kit for vaccinating an animal, preferably a pig, against a disease associated with swine streptococcal infection, optionally including instructions for use.   Bacteria or cultures deposited on August 19, 2015 at the Centralbureau voor Schimmelcultures as "CBS140425 Streptococcus pneumoniae ΔFolT mutant".   Bacteria or culture thereof deposited on August 25, 2017 at the Westerdikk Fungal Biodiversity Institute as “CBS143192 Streptococcus pyococcus ΔFolT2 mutant”.   A method of reducing bacterial toxicity comprising reducing the ability of a bacterium to transport folic acid.   17. A method according to claim 16, wherein the bacterium has the capacity for novel folic acid synthesis.   18. The method of claim 16 or 17, comprising attenuating bacterial folate substrate binding protein (FolT) expression and / or function.   The method according to any one of claims 16 to 18, wherein the toxicity is attenuated by performing mutation, deletion or insertion in a bacterial fltT gene.   20. The method of claim 19, wherein the toxicity is attenuated by mutating, deleting or inserting into the bacterial DNA encoding the promoter of the folT gene.   21. The method according to claim 19 or 20, wherein the toxicity is attenuated by mutating, deleting, or inserting into a bacterial DNA encoding a transmembrane region of the folate substrate binding protein FolT.   The method according to any one of claims 19, 20, or 21, wherein the toxicity is attenuated by mutating, deleting or inserting in FolT encoding a bacterial DNA region encoding a region important for substrate binding. .   A ΔFolT mutant deposited on August 19, 2015 at the Centralbureau voor Schumelculures as Fermicutes, preferably Streptococcus, more preferably Streptococcus pneumoniae, more preferably “CBS140425 Streptococcus pneumoniae ΔFolT mutant”, 23. The method according to any one of claims 16 to 22, which is most preferably a ΔFolT mutant deposited on August 25, 2017 at the Westerijk Fungal Biodiversity Institute as a “CBS143192 Streptococcus pneumoniae ΔFolT2 mutant”.   24. A bacterium with reduced toxicity obtained or obtained by the method according to any one of claims 16 to 23.   25. A bacterial culture according to claim 24.   26. A composition comprising the bacterium of claim 24 or the culture of claim 25.   25. An immunogenic composition comprising the bacterium of claim 24 or the culture of claim 25.   28. Use of a culture according to claim 25 or a composition according to claim 26 or 27 for the production of a vaccine.   A vaccine comprising a bacterium according to claim 24 or a culture according to claim 25, a composition according to claim 26 or 27.   Selecting a parent bacterial strain that is generally capable of folate transport and folate synthesis, and selecting a bacterium from said parent strain for mutation, deletion or insertion in DNA encoding the bacterial folate substrate binding protein; Selecting a bacterium that grows in vitro to the same rate as the parent strain but has an ability to grow to a lower rate in vivo than the parent strain.   Selecting a parent bacterial strain generally capable of folate transport and folate synthesis, and transforming the bacterium from said parent strain by mutating, deleting or inserting in the DNA encoding the bacterial folate substrate binding protein A method of producing a bacterium comprising the steps of: selecting a bacterium that grows in vitro to the same rate as the parental strain but has an ability to grow to a lower rate in vivo than the parental strain.   A bacterium obtainable or obtained by the method according to claim 30 or 31.   33. Bacteria according to claim 32, which can be classified as Pharmicutes, preferably streptococci, more preferably swine streptococci.   34. A bacterial culture according to claim 32 or 33.   35. A composition comprising the bacterium of claim 32 or 33 or the culture of claim 34.   An immunogenic composition comprising the bacterium of claim 32 or 33 or the culture of claim 34.   Use of the composition according to claim 35 or 36 for the production of a vaccine.   A vaccine comprising the bacterium according to claim 32 or 33 or the culture according to claim 34 or the composition according to claim 35 or 36.